Chitin is a linear homopolymer of .beta.-(1,4)-linked N-acetylglucosamine residues. This polysaccharide is second only to cellulose as the most abundant organic substance. The exoskeleton of arthropods is composed of chitin. In addition, fungi and other parasites contain chitin in their outer cell wall, where it serves important structural and protective roles. Disruption of the fungal cell wall and membrane has been a useful therapeutic strategy against fungi and parasites. For example, Amphotericin B and fluconazole exert their anti-fungal activity by affecting membrane steroids. Despite the existence of anti-fungal therapeutics, fungal infections of humans have increasingly become responsible for life-threatening disorders. See, Georgopapadakou et al., Trends Microbiol., 3:98-104 (1995). The fungal species and parasites responsible for these diseases are mainly Candida, Aspergillus, Cryptococcus, Histoplasma, Coccidioides and Pneumocystis. These pathogens are particularly dangerous in immunocompromised individuals, such as patients with AIDS, patients undergoing chemotherapy, and immunosuppressed organ transplant patients.
Chitin can be degraded by the enzyme chitinase. Chitinase enzymes are found in plants, microorganisms, and animals. Bacterial chitinase helps to provide a carbon source for bacterial growth. Insects produce chitinase to digest their cuticle at each molt. In plants, chitinase is thought to provide a protective role against parasitic fungi. Chitinases have been cloned from numerous bacterial [e.g., Serratia marcescens, Jones et al., EMBO J., 5:467-473 (1986)], plant [e.g., tobacco, Heitz et al., Mol. Gen. Genet., 245:246-254 (1994)], and insect [e.g., wasp, Krishnan et al., J. Biol. Chem., 269:20971-20976 (1994)] species and have been categorized into two distinct families, designated family 18 and family 19, based on sequence similarities [Henrissat and Bairoch, Biochem, J. 293:781-788 (1993)]. Although the catalytic region of the enzymes in family 18 is largely conserved across numerous species, there is very limited sequence similarity across species for the chitin-binding domain. The only feature common to several family 18 chitin-binding domains is the presence of multiple cysteine residues.
Several proteins with low homology to bacterial, insect, and plant chitinases (less than 40% amino acid identity) have been identified in mammals, such as human cartilage gp-39 (C-gp39) [Hakala et al., J. Biol. Chem., 268:25803-25810 (1993)], human glycoprotein YKL-40 [Johansen et al., Eur. J. Cancer, 31A: 1437-1442 (1995)], oviduct-specific, estrogen-induced protein from sheep [DeSouza et al., Endocrinology, 136:2485-2496 (1995)], cows and humans; and a secretory protein from activated mouse macrophages [Chang et al., Genbank M94584]. However, chitin-degrading activity has not been reported for these proteins. The function of these proteins is not known, but they have been postulated to be involved in tissue remodeling. Hakala et al., supra, report that C-gp39 is detectable in synovial and cartilage specimens from rheumatoid arthritis patients, but not from normal humans. Recklies et al., Arthritis Rheumatism, 36(9 SUPPL.):S190 (1993) report localization of the C-gp39 protein to a distinct population of cells in the superficial layers of cartilage. Johansen et al., supra, report that measurements of YKL-40 serum levels are of value as a potential prognostic marker for the extent of metastatic disease and survival of patients with recurrent breast cancer.
Escott et al., Infect. Immun., 63:4770-4773 (1995) demonstrated chitinase enzymatic activity in human leukocytes and in human serum. Overdijk et al., Glycobiology, 4:797-803 (1994) described isolation of a chitinase (4-methylumbelliferyl-tetra-N-acetylchitotetraoside hydrolase) from human serum and rat liver. Renkema et al., J. Biol. Chem., 270:2198-2202 (February 1995) prepared a human chitotriosidase from the spleen of a Gaucher disease patient. Their preparation exhibited chitinase activity and the article reports a small amount of amino acid sequence of the protein component of the preparation (22 amino terminal residues and 21 residues of a tryptic fragment). The function of human chitinase is also unknown, but a relationship with the pathophysiology of Gaucher disease is proposed in the article. A later publication by the same group [Boot et al., J. Biol. Chem., 270(44):26252-26256 (November 1995)] describes the cloning of a human macrophage cDNA encoding a product that exhibits chitinase activity. The partial amino acid sequence reported by the group in their February 1995 article matches portions of the deduced amino acid sequence of the human macrophage cDNA product. See also International Patent Publication No. WO 96/40940, which reports two distinct human chitotriosidase cDNAs encoding a 50 kD and a 39 kD product, both of which were fully enzymatically active. Renkema et al., Eur. J. Biochem., 244:279-285 (1997) reported that human chitinase is initially produced in macrophages as a 50 kD protein that is in part processed into a 39 kD form that accumulates in lysozymes, and also reported that alternative splicing generates a distinct human chitinase mRNA species encoding a 40 kD chitinase. Both the 39 kD and 40 kD isoforms appeared to be C-terminally truncated and displayed full chitinase enzymatic activity but bound chitin poorly.
In view of the increasing incidence of life-threatening fungal infection in immunocompromised individuals, there exists a need in the art to identify new materials and methods useful for diagnosing and treating fungal infections.